Biochemical Characterization of a Novel Monospecific Endo-β-1,4-Glucanase Belonging to GH Family 5 From a Rhizosphere Metagenomic Library

Cellulases have a broad range of different industrial applications, ranging from food and beverages to pulp and paper and the biofuels area. Here a metagenomics based strategy was used to identify the cellulolytic enzyme CeIRH5 from the rhizosphere. CelRH5 is a novel monospecific endo-beta-1,4-glucanase belonging to the glycosyl hydrolase family 5 (GH5). Structural based modeling analysis indicated that CelRH5 is related to endo-beta-1,4-glucanases derived from thermophilic microorganisms such as Thermotoga maritima, Fervidobacterium nodosum, and Ruminiclostridium thermocellum sharing 30-40% amino acid sequence identity. The molecular weight of the enzyme was determined as 40.5 kDa. Biochemical analyses revealed that the enzyme displayed good activity with soluble forms of cellulose as a substrate such as ostazin brilliant red hydroxyethyl cellulose (OBR-HEC), carboxymethylcellulose (CMC), hydroxyethyl cellulose (HEC), and insoluble azurine cross-linked hydroxyethylcellulose (AZCL-HEC). The enzyme shows highest enzymatic activity at pH 6.5 with high pH tolerance, remaining stable in the pH range 4.5-8.5. Highest activity was observed at 40 degrees C, but CelRH5 is psychrotolerant being active and stable at temperatures below 30 degrees C. The presence of the final products of cellulose hydrolysis (glucose and cellobiose) or metal ions such as Na+, K+, Li+, and Mg2+, as well as ethylenediaminetetraacetic acid (EDTA), urea, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME) or glycerol, did not have a marked effect on CelRH5 activity. However, the enzyme is quite sensitive to the presence of 10 mM ions Zn2+, Ni2+, Co2+, Fe3+ and reagents such as 1 M guanidine HCl, 0.1% sodium dodecyl sulfate (SDS) and 20% ethanol. Given that it is psychrotolerant and retains activity in the presence of final cellulose degradation products, metal ions and various reagents, which are common in many technological processes; CelRH5 may be potential suitability for a variety of different biotechnological applications.